The success of the Edmonton protocol, demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. Despite the progress in islet isolation procedures, a single donor transplant does not provide enough islets to attain insulin independence. There is evidence that significant loss of islet cells takes place during isolation due to the induction of apoptosis, and that the reduction of isolation-induced apoptosis can improve the success rate of islet transplantation. The goal of this proposal is to address the needs for reduced apoptosis and improved viability of islets. We propose to develop novel transduction methods that would allow manipulation of islets to reduce apoptosis. New methods allowing the direct transfer of proteins to living cells using protein transduction domains (PTDs) appear particularly promising for this application. In particular, proteins fused to an 11-amino acid PTD from the human immunodeficiency virus TAT protein, readily diffuse across membranes and are efficiently transduced into virtually all cell types. The expression as well as the biological function of the TAT fusion protein are temporary without permanent modification of the sensitivity of the cell to apoptosis, thus avoiding undesirable long-term effects. Our central hypothesis is that the transduction of the pancreas with TAT PTD antiapoptotic fusion proteins prior to the islet isolation, will reduce apoptosis, induced as a consequence of isolation procedures and enhance the viability of islets. To test this hypothesis, we will define methods for ex vivo delivery of several TAT anti- apoptosic fusion proteins (TAT-BCL-XL, TAT-PEA-15, TAT-HO-1) to mouse pancreas prior to islet isolation. We will then investigate the molecular and morphological events during apoptosis and the efficacy of these TAT antiapoptotic fusion proteins to prevent apoptosis in isolated mouse islets. In order to fully understand the mechanism of the TAT antiapoptotic fusion proteins we will study in vivo the efficacy to improve the ability of transplanted islets to reverse diabetes. The success of these studies will lay the groundwork for further studies to evaluate the potential of this novel approach in human islet isolation.